In vertebrates, numerous tissues and organs are derived in whole, or in part, from the mesoderm, including the skeletal muscles, heart, skeleton and kidney. A complex network of genes regulates the progressive specification of these various mesoderm derivatives. In Xenopus, the mesoderm forms from a band of cells around the equator of the early gastrula embryo. In models of mesoderm patterning in Xenopus, the Spemann organizer has figured prominently, and is thought to specify mesodermal fates through its secretion of inducing factors. We have recently shown that a Spemann organizer-independent pathway establishes a pattern along the animal-vegetal axis of the mesoderm by the mid-gastrula stage. Two distinct domains can be observed in this axis. The animal domain expresses the gene brachyury and appears to be fated to become notochord and somite, while the vegetal domain is fated to become ventrolateral mesoderm. The goal of this proposal is to further elucidate the mechanisms that act to establish patterning along the animal/vegetal axis. In particular, we will investigate the role of the nodal growth factors in the induction of ventral mesoderm. Our preliminary results indicate that Xenopus nodal-related-2 (Xnr2)-expressing cells are restricted to the presumptive ventral mesoderm in the gastrula embryo. Lineage tracing experiments proposed here are aimed at rigorously testing the fate of these cells. Conclusively establishing the fates of cells within the early presumptive mesoderm, and correlating them with restricted gene expression patterns, is an essential first step in elucidating the mechanism and timing of specification of fates. We have also hypothesized that nodals plays an essential role in the induction of ventral mesoderm, and we will examine the activity of nodals in the gastrula embryo by use of dominant negative constructs and inhibitory binding proteins. Finally, our preliminary results show that the combination of Xnr2 signaling and low FGF activity, such as exists in the vegetal marginal zone, makes ectoderm explants differentiate as blood, a ventral mesoderm derivative, in response to bone morphogenetic proteins (BMP). We will use subtraction and expression cloning approaches to identify genes downstream of Xnr2 signaling that are responsible for altering the competence of ectoderm explants to BMP signaling. Genes that are identified in this screen will be further characterized to place them in the pathway of ventrolateral mesoderm induction.